Tubular graft construct

ABSTRACT

Described are medical devices which are or can be used to form tubular medical devices, and related methods. Preferred devices include tubular grafts of biomaterial having lumen walls which present no seam edge that traverses the entire length of the lumen, illustratively including devices having lumen walls which have a discontinuous seam presenting multiple seam edges. Such a device may include a tubular structure formed by inserting a plurality of extensions of a biomaterial sheet through a plurality of corresponding apertures of the sheet.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/068,212, filed Feb. 6, 2002, which is a continuation of InternationalPatent Application Serial No. PCT/US00/21546, filed Aug. 7, 2000designating the United States and published in English, which claims thebenefit of U.S. Provisional Patent Application Ser. No. 60/147,647 filedAug. 6, 1999, each of which is incorporated herein by reference in itsentirety.

TECHNICAL FIELD

The invention relates generally to a medical device, and moreparticularly, to a tubular graft biomaterial device.

BACKGROUND OF THE INVENTION

It has become common to treat a variety of medical conditions byintroducing an implantatile medical device into the alimentary,circulatory, coronary, urological, renal, and other organ systems. Forexample, coronary vessels via delivery catheters, such as ballooncatheters.

In the case of aneurysm treatment, an aneurysm is caused by a weakeningof the vessel wall, which causes an invagination of the vessel wall.Blood flow is inhibited at the neck of the aneurysm due to turbulencecaused by blood entering and exiting the lumen of the aneurysm. Currentmedical treatment of aneurysms include the use of metal coils, such asthe FDA approved Gugliemi Detachable Coil, inserted into the lumen ofthe aneurysm. However, this platinum coil is relatively soft and doesnot provide a complete packing of the aneurysm lumen. It is not uncommonfor the aneurysm to re-canalize, enlarge, and even rupture. The heavymetal used in the coils provide the necessary radiographic visualizationto ensure that the coils are localized properly and whether, during asubsequent examination, the coils remain in the situs.

However, there are problems associated with using synthetic materials,which include thrombus formation, immune response leading to rejection,and undesired occlusion of the vessel. Therefore, a better material forimplantation in any application, such as coronary, vascular, body wallrepair, orthopaedic, tissue graft, dermal, and other industries isneeded. One such material is a newly discovered biomaterial comprisingtissue mucosa, tissue serosa, or tissue submucosa.

Tissue implants in a purified form and derived from collagen-basedmaterials have been manufactured and disclosed in the literature.Cohesive films of high tensile strength have been manufactured usingcollagen molecules or collagen-based materials. Aldehydes, however, havebeen generally utilized to cross-link the collagen molecules to producefilms having high tensile strengths. With these types of materials, thealdehydes may leech out of the film, e.g. upon hydrolysis. Because suchresidues are cytotoxic, the films are poor tissue implants.

Other techniques have been developed to produce collagen-based tissueimplants while avoiding the problems associated with aldehydecross-linked collagen molecules. One such technique is illustrated inU.S. Pat. No. 5,141,747 wherein the collagen molecules are cross-linkedor coupled at their lysine epsilon amino groups followed by denaturingthe coupled, and preferably modified, collagen molecules. The discloseduse of such collagen material is for tympanic membrane repair. Whilesuch membranes are disclosed to exhibit good physical properties and tobe sterilized by subsequent processing, they are not capable ofremodeling or generating cell growth or, in general, of promotingregrowth and healing of damaged or diseased tissue structures.

In general, researchers in the surgical arts have been working for- manyyears to develop new techniques and materials for use as implants toreplace or repair damaged or diseased tissue structures, for example,blood vessels, aneurysms, muscle, ligaments, tendons and the like. It isnot uncommon today, for instance, for an orthopedic surgeon to harvest apatellar tendon of autogenous or allogenous origin for use as areplacement for a torn cruciate ligament. The surgical methods for suchtechniques are known. Further, it has been common for surgeons to useimplantable prostheses formed from plastic, metal and/or ceramicmaterial for reconstruction or replacement of physiological structures.Despite their wide use, surgical implanted prostheses present manyattendant risks to the patient.

Researchers have also been attempting to develop satisfactory polymer orplastic materials to serve as functional tissue structures and/or otherconnective tissues, e.g., those involved in hernia and joint dislocationinjuries. It has been discovered that it is difficult to provide atough, durable plastic material which is suitable for long, termconnective tissue replacement. The tissues surrounding the plasticmaterial can become infected and difficulties in treating suchinfections often lead to the failure of the implant or prostheses.

As mentioned above, various collagen-based materials have also beenutilized for the above-mentioned tissue replacements; however, thesematerials either did not exhibit the requisite tensile strength or alsohad problems with infection and other immunogenic responses,encapsulation, or had other problems. In a related patent, U.S. Pat. No.5,372,821, it is disclosed that a submucosa collagenous biomaterial maybe sterilized by conventional techniques, e.g., aldehyde tanning,propylene oxide, gamma radiation and peracetic acid. No specificprocessing steps are disclosed except that the submucosa layer is firstdelaminated from the surrounding tissue prior to sterilizationtreatment.

Some materials considered desirable are biological materials(biomaterials) from autogenous, allogenous, or xenogeneic(heteroplastic) sources. Biomaterials are desirable as they can bemalleable and less likely to be rejected as foreign. One suchbiomaterial is collagen. Collagen is a protein molecule that comes inmany types. For example, collagen Type I constitutes a significantamount of the collagen in the body. Type I is a heterotrimeric molecule,has a helical configuration, and is characterized by a Glycine-X-Y aminoacid repeating sequence. Due to its abundance in the human body,collagen is being examined for its uses in medical treatment.

One of the problems associated with biomaterials includes leakage orseepage from the tubular graft. Particularly, a graft made by suturingtwo ends of a flat sheet together cause holes to extend from the lumenalside to the outside, thus providing small channels for lumenal fluid toseep. A reduced seepage biomaterial construct is desired and would bewell-received.

Problems associated with synthetic grafts are well-documented. Forexample, it is known that the mechanical properties of synthetic graftsdegrade over time, as described in Vinard et al, Stability ofPerformances of Vascular Prostheses Retrospective Study of 22 Cases ofHuman Implanted Prostheses, Vol. 22(7) Jour. of Biomedical MaterialsResearch pg. 633-648 (July 1988); and Manfredi et al., VascularProstheses, vol. 12(3) Emergency Medicine Clinics of North America pg.657-77 (August 1994). A graft that gets stronger over time is moredesirable than one that degrades over time.

One other problem associated with synthetic or biomaterial graftsinclude the occlusion of the lumen itself. Often times, the graftmaterials come loose or the layers comprising the graft separate, thuscausing material to hang into the lumen. This debris causesthrombogenesis and reduces patency of the graft. If the thrombus were todislodge, disastrous effects will soon follow.

SUMMARY OF THE INVENTION

The foregoing problems are solved and a technical advance is achieved ina newly discovered graft construct comprising a biomaterial. Oneexemplary biomaterial is a newly discovered collagenous material calledtela submucosa, which has been shown to be a remarkable biomaterial thatpromotes remodeling of the surrounding tissue, such as cellularinvasion, host incorporation, and absorption of the tela submucosamaterial into the local tissue. One exemplary tela submucosa is smallintestine submucosa (SIS). Furthermore, SIS has been shown to beacellular, strong, and exhibit a sidedness in that it has a differentialporosity of its mucosal and serosal sides. Highly purified SIS also doesnot trigger any negative immune system response as evidence suggeststhat it has no viral activity when checking for enveloped,non-enveloped, DNA, and RNA virus. Studies also show that SIS increasesthe Th-2 immune response by increasing the production of interleukin-10over interferon-y, which indicates that the immune response is moreaccommodation than rejection. Due to these and other properties, SISmakes for an excellent implantable biomaterial for use in multipleindustries.

While collagenous biomaterials are known in the industries, none speakto the collagenous biomaterial being reduced in seepage or leakageproperties. As mentioned above a tubular graft comprising a biomaterialthat has its manufacturing holes sealed or covered over providesincreased structural integrity and reduced seepage from the tube.

In accordance with the present invention, provided is a collagenousimplantable biomaterial, such as tela submucosa, such as small intestinesubmucosa (SIS) that is further advantageous in that it is created insuch a manner to reduce seepage. Further provided in accordance with thepresent invention is the biocompatibility of the present device overother presently available biomaterials. The present invention furthercomprises a collagenous biomaterial that has an endotoxin level of lessthan 12 endotoxin units per gram. Advantageously, the present inventionpermits capitalization of the newly discovered collagenous biomaterialand its biotropic properties to be combined with vessel graftconstructs. This permits further industrial application of the disclosedmedical device.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents an end view of the biomaterial.

FIG. 2 represents a top down view of the flat biomaterial.

FIGS. 3 and 3A represent other embodiments of the present inventionincluding retainers.

FIG. 4 represents extensions inserted into the apertures.

FIG. 5 represents a side view of the extensions inserted into theapertures, showing multiple extensions.

FIG. 6 represents a side view of one extension inserted into theaperture.

FIG. 7 represents a side view of an intermediate layer disposed over theaperture.

FIG. 8 represents side view of an intermediate layer disposed betweenthe aperture and the surplus biomaterial.

FIG. 9 represents another embodiment of the present invention.

FIG. 10 represents an outer layer disposed over the graft construct.

FIG. 11 represents a series of retainers on a graft of the invention.

FIG. 12 provides a cut-out representing a single retainer of theinvention.

FIG. 13 represents a retainer inserted through a corresponding aperturein a graft of the invention.

FIGS. 14-18 represent additional graft constructs of the invention.

DETAILED DESCRIPTION

In the discussions herein, a number of terms are used. In order toprovide a clear and consistent understanding of the specification andclaims, the following definitions are provided.

Bioburden—refers to the number of living microorganisms, reported in 30colony-forming units (CFU), found on and/or in a given amount ofmaterial. Illustrative microorganisms include bacteria, fungi and theirspores.

Disinfection—refers to a reduction in the bioburden of a material.

Sterile—refers to a condition wherein a material has a bioburden suchthat the probability of having one living microorganism (CFU) on and/orin a given section of the material is one in one-million or less.

Pyrogen—refers to a substance which produces febrile response afterintroduction into a host.

Endotoxin—refers to a particular pyrogen which is part of the cell wallof gram-negative bacteria. Endotoxins are continually shed from thebacteria and contaminate materials.

Purification—refers to the treatment of a material to remove one or morecontaminants which occur with the material, for instance contaminantswith which the material occurs in nature, and/or microorganisms orcomponents thereof occurring on the material. Illustratively, thecontaminants may be those known to cause toxicity, infectivity,pyrogenicity, irritation potential, reactivity, hemolytic activity,carcinogenicity and/or immunogenicity.

Biocompatibility—refers to the ability, of a material to pass theappropriate or relevant biocompatibility test or tests set forth inInternational Standards Organization (ISO) Standard No. 10993, or theU.S. Pharmacopeia (USP) 23, or the U.S. Food and Drug Administration(FDA) blue book memorandum No. G95-1, entitled “Use of InternationalStandard ISO-10993, Biological Evaluation of Medical Devices Part-1:Evaluation and Testing.” Typically, these tests assay as to a material'stoxicity, infectivity, pyrogenicity, irritation potential, reactivity,hemolytic activity, carcinogenicity and/or immunogenicity. Abiocompatible structure or material when introduced into a majority ofpatients will not cause an adverse reaction or response. In addition, itis contemplated that biocompatibility can be effected by othercontaminants such as prions, surfactants, oligonucleotides, and otherbiocompatibility effecting agents or contaminants.

Contaminant—refers to an unwanted substance on, attached to, or within amaterial. This includes, but is not limited to: bioburden, endotoxins,processing agents such as antimicrobial agents, blood, blood components,viruses, DNA, RNA, spores, fragments of unwanted tissue layers, cellulardebris, and mucosa.

Tela submucosa—refers to a layer of collagen-containing connectivetissue occurring under the mucosa in most parts of the alimentary,respiratory, urinary, integumentary, and genital tracts of animals.

Includes—refers to a list of items included but does not limit the list,e.g., such as but is not limited to, the following items . . . .

With reference to FIGS. 1 and 2, the graft 10, initially starts off as aflat sheet. The flat sheet generally comprises a first biomaterial 12such as, but not limited to, at least one of a submucosal tissue,mucosal tissue, serosal tissue, collagen, partially collagenousbiomaterial, elastin, polytetrafluoroethylene, polyester, stainlesssteel, DACRON(R), ORLON(R), FORTISAN(R), nylon, polypropylene,polyglactin 910, polyglycolic acid, pericardium, dura tissue, facialata, a biocompatible material, a synthetic material, polymers,polypyrramidole, co-polymers, and/or any combination or part thereof.One such first biomaterial 12 includes collagenous biomaterial, such astissue mucosa or tela submucosa which also further includes a tissuesubmucosa, which further includes a small intestine submucosa (SIS). Itis understood that any reference to tela submucosa also includes tissuemucosa or tissue serosa. Tela submucosa is a multi-laminate structure,comprising the tunica submucosa, lamina muscularis mucosa, and thestratum compactum. Tela submucosa has biotropic agents comprising atleast one of a proteoglycan glycosaminoglycan, and growth factor. Thetela submucosa can be made using the techniques described in Cook etal., WIPO Publication WO 98/22185, dated 28 May 1998, which is thepublished application of PCT/US97/14855, the disclosure of which is setforth below. Tela submucosa is a decellularized or acellular tissue,which means it is devoid of intact viable cells, although some cellcomponents may remain in the tissue. All types of collagenous materials,as are any type of biocompatible synthetic materials, are contemplatedherein. For example, included herein are: gastric submucosa as describedin PCT/US97/22729, published as WO 98/26291; liver tissue as describedin PCT/US97/22727, published as WO 98/25637; stomach tissue as describedin PCT/US97/23010, published as WO 98/25636; and urinary tissue asdescribed in U.S. Pat. No. 5,554,389, issued to Badylak et al., andbovine serosa.

One type of biomaterial is mucosa, such as tela submucosa, and as withmany animal tissues, is generally aseptic in its natural state, providedthe human or animal does not have an infection or disease. This isparticularly the case since the tela submucosa is an internal layerwithin the alimentary, integumentary, respiratory, urinary, and genitaltracts of animals. Accordingly, it is generally not exposed to bacteriaand other cellular debris such as the epithelium of the intestinaltract. One feature of the present invention is the discovery that bydisinfecting the source tissue for the tela submucosa prior todelamination, the aseptic state of the tela submucosa layer can bepreserved or substantially preserved, particularly if the delaminationprocess occurs under sterile conditions.

In particular, it has been discovered that disinfecting the telasubmucosa source, followed by removal of a purified biomaterialincluding the tela submucosa, e.g. by delaminating the tela submucosafrom the tunica muscularis and the tunica mucosa; minimizes the exposureof the tela submucosa to bacteria and other contaminants. In turn, thisenables minimizing exposure of the isolated tela submucosa biomaterialto disinfectants or sterilants if desired, thus substantially preservingthe inherent biochemistry of the tela submucosa and many of the telasubmucosa's beneficial effects.

A tela submucosa implantable collagen biomaterial according to thepresent invention can, as indicated above, be obtained from thealimentary, respiratory, urinary, integumentary, or genital tracts ofanimals. Preferably, the tele submucosa tissues, which arecollagen-based and thus predominantly collagen, are derived from thealimentary tract of mammals, such as cows, sheep, dogs, and mostpreferably from the intestinal tract of pigs. A most preferred source ofwhole small intestine is harvested from mature adult pigs weighinggreater than about 450 pounds. Intestines harvested from healthy,non-diseased animals will contain blood vessels and blood supply withinthe intestinal tract, as well as various microbes such as E. colicontained within the lumen of the intestines. Therefore, disinfectingthe whole intestine prior to delamination of the tele submucosasubstantially removes these contaminants and provides a preferredimplantable tele submucosa tissue which is substantially free of bloodand blood components as well as any other microbial organisms, pyrogensor other pathogens that may be present. In effect, this procedure isbelieved to substantially preserve the inherent aseptic state of thetele submucosa, although it should be understood that it is not intendedthat the present invention be limited by any theory.

It is also desirable that the first biomaterial 12, such as thecollagenous biomaterial according to the present invention besubstantially free of any antiviral agents or any antimicrobial typeagents which can affect the biochemistry of the biomaterial and itsefficacy upon implantation. In the past, one method of treating suchtissue material is to rinse the delaminated tissue in saline and soak itin an antimicrobial agent, for example, as disclosed in U.S. Pat. No.4,956,178. While such techniques can optionally be practiced withisolated collagenous mucosa or submucosa of the present invention,preferred processes according to the present invention avoid the use ofantimicrobial agents and the like which can not only affect thebiochemistry of the collagenous biomaterial but also can beunnecessarily introduced into the tissues of the patient.

As discussed above, it has been discovered that a highly pure form of animplantable tela submucosa collagen biomaterial can be obtained by firstdisinfecting a tela submucosa source prior to removing a purifiedcollagen biomaterial including the tela submucosa layer, e.g. bydelaminating the tela submucosa source. It has also been discovered thatcertain processing advantages as well as improved properties of theresultant tela submucosa layer are obtained by this process, includinggreater ease in removing attached tissues from the submucosa layer, anda characteristic, low contaminant profile.

Processes of the invention desirably involve first rinsing the telasubmucosa source one or more times with a solvent, suitably water. Therinsing step is followed by treatment with a disinfecting agent. Thedisinfecting agent is desirably an oxidizing agent. Preferreddisinfecting agents are peroxy compounds, preferably organic peroxycompounds, and more preferably peracids. Such disinfecting agents aredesirably used in a liquid medium, preferably a solution, having a pH ofabout 1.5 to about 10, more preferably a pH of about 2 to about 6, andmost preferably a pH of about 2 to about 4. In methods of the presentinvention, the disinfecting agent will generally be used underconditions and for a period of time which provide the recovery ofcharacteristic, purified submucosa matrices as described herein,preferably exhibiting a bioburden of essentially zero and/or essentialfreedom from pyrogens. In this regard, desirable processes of theinvention involve immersing the tissue source (e.g. by submersing orshowering) in a liquid medium containing the disinfecting agent for aperiod of at least about 5 minutes, typically in the range of about 5minutes to about 40 hours, and more typically in the range of about 0.5hours to about 5 hours.

A preferred peroxy disinfecting agent is hydrogen peroxide. Theconcentration of hydrogen peroxide can range from about 0.05% to 30% byvolume. More preferably the hydrogen peroxide concentration is fromabout 1% to 10% by volume and most preferably from about 2% to 5% byvolume. The solution can or can not be buffered to a pH from about 5 to9. More preferably the pH is from about 6 to 7.5. These concentrationscan be diluted in water or in an aqueous solution of about 2% to about30% by volume alcohol. Most preferably the alcohol is ethanol. Thesolution temperature can range from about 15 to 50° C. More preferablythe solution temperature is from about 20 to 40° C. Most preferably, thesolution temperature is from about 32 to 37° C. The exposure time canrange from about 10 to 400 minutes. Preferably, the exposure time isfrom about 120 to 240 minutes. More preferably, the exposure time isfrom 180 to 210 minutes.

A preferred organic peroxide disinfecting agent is perpropionic acid.The concentration of perpropionic acid can range from about 0.1% to0.10% by volume. More preferably the perpropionic acid concentration isfrom about 0.1% to 1.0% by volume and most preferably from about 0.2% to0.5% by volume. These concentrations of perpropionic acid can be dilutedin water or in an aqueous solution of about 2% to about 30% by volumealcohol. Most preferably the alcohol is ethanol. The tela submucosatissue source can be exposed to the organic peroxide solution forperiods from about 15 minutes to about 40 hours, and more typically inthe range of about 0.5 hours to about 8 hours. Other peroxy disinfectingagents are suitable for use as described in “Peroxygen Compounds”, S.Block, in Disinfection, Sterilization and Preservation, S. Block,Editor, 4th Edition, Philadelphia, Lea & Febiger, pp. 167-181, 1991; and“Disinfection with peroxygens”, M. G. C. Baldry and J. A. L. Fraser, inIndustrial Biocides, K. Payne, Editor, New York, John Wiley and Sons,pp. 91-116, 1988.

Another oxidizing disinfecting agent is chlorhexidine(1,6-di(4-chlorophenyldiguanido)hexane) in its digluconate form. Theconcentration of chlorhexidine digluconate can range from about 0.1% to15% by weight. More preferably, the chlorhexidine digluconateconcentration is from about 0.1% to 2% by weight and most preferablyfrom about 0.2% to 5% by weight. The solution can or can not be bufferedto a pH from about 5 to 8. More preferably the pH is from about 5.5 to7. These concentrations can be diluted in water or in an aqueoussolution of about 2% to about 20% by volume alcohol. Most preferably thealcohol is ethanol at a concentration of about 5% to 10%. The solutiontemperature can range from about 15 to 30° C. The exposure time canrange from about 10 to 400 minutes. More preferably the exposure time isfrom about 30 to 60 minutes. Other chlorine agents are described in“Chlorhexidine”, G. W. Denton, in Disinfection, Sterilization andPreservation, S. Block, Editor, 4th Edition, Philadelphia, Lea &Febiger, pp. 274-289, 1991.

In preferred preparative processes, a peracid or other disinfectingagent can be dissolved in a dilute aqueous alcohol solution, preferablywherein the alcohol has from 1 to about 6 carbon atoms, and wherein thealcohol can generally comprise from about 1% to about 30% by volume ofthe solution. More preferred alcohols for use in the invention areselected from the group consisting of ethanol, propanols and butanols.Ethanol is a preferred alcohol for these purposes.

When a peracid is used in the disinfection, it is preferably selectedfrom the group consisting of peracetic acid, perpropionic acid orperbenzoic acid. Peracetic acid is the most preferred disinfectingagent. The peracetic acid is preferably diluted into about a 2% to about10% by volume alcohol solution. The concentration of the peracetic acidcan range, for example, from about 0.05% by volume to about 1.0% byvolume. Most preferably the concentration of the peracetic acid is fromabout 0.1% to about 0.3% by volume. Hydrogen peroxide can also be usedas a disinfecting agent. Alternatively, or in addition, the telesubmucosa tissue source, e.g. from small intestine, can be disinfectedutilizing disinfecting agents such as glutaraldehyde, formalin and thelike, which are also known for their ability to introduce substantialcrosslinking into collagen matrices, in contrast to the action of otherdisinfecting agents such as peracids which can be used to disinfectwithout introducing such crosslinking. Additionally, the tele submucosasource can be treated with radiation, e.g., gamma radiation, forpurposes of disinfection.

Variations on the disinfection process can also include the following:

1. Intestine is treated with 0.2% peracetic acid, 5% ethanol solution ata ratio of 10:1 solution to intestine ratio by weight. Solution has a pHof 2.6. Solution and intestine are vigorously mixed for two hours.

2. Intestine is treated with 1% peracetic acid, 25% ethanol solution ata ration of 5:1 solution to intestine ratio by weight. Solution has a pHof 2. Solution and intestine are vigorously mixed for one hour.

3. Intestine is treated with 1% peracetic acid, 15% ethanol, and 10%hydrogen peroxide solution at a ratio of 5:1 solution to intestine ratioby weight. Solution and intestine are vigorously mixed for one hour.

4. Whole small intestine is rinsed four times with high purity water for15 minutes. The intestine is then subjected to 1.5 MRAD Electron Beamradiation.

5. Whole small intestine is rinsed four times with high purity water for15 minutes. Lengthwise along a conveyor belt, the intestine is subjectedto high-intensity pulsed light which disinfects the intestine.

Following the treatment as described above, the tela submucosa layer isdelaminated from its source, e.g., whole intestine, cow uterus and thelike. It has been found that by following thispost-disinfection-stripping procedure, it is easier to separate the telasubmucosa layer from the attached tissues, e.g. at least from attachedtunica muscularis tissue, as compared to stripping the tele submucosalayer prior to disinfection. Moreover it has been discovered that theresultant tele submucosa layer in its most preferred form exhibitssuperior histology, in that there is less attached tissue and debris onthe surface compared to a tele submucosa layer obtained by firstdelaminating the tele submucosa layer from its source and thendisinfecting the layer. Moreover, a more uniform tele submucosa tissuecan be obtained from this process, and a tele submucosa having the sameor similar physical and biochemical properties can be obtained moreconsistently from each separate processing run. Importantly, a highlypurified, substantially sterile tele submucosa is obtained by thisprocess. The stripping of the tela submucosa source is preferablycarried out by utilizing a disinfected or sterile casing machine, toproduce a tele submucosa which is substantially sterile and which hasbeen minimally processed. A suitable casing machine is the Model 3-U-400Stridhs Universal Machine for Hog Casing, commercially available fromthe AB Stridhs Maskiner, GOtoborg, Sweden. Therefore, the measuredbioburden levels are minimal or substantially zero. Of course, othermeans for delaminating the tele submucosa source can be employed withoutdeparting from the present invention, including for example those meanswell known in the art, including delaminating by hand.

It has also been discovered that more preferred processes according tothe present invention, not only will eliminate or significantly reducecontaminants contained in the tela submucosa collagen biomaterial, butalso will produce a tissue which exhibits no substantial degradation ofphysical and mechanical properties, e.g., differential porosity (i.e.wherein one side of the submucosa layer has greater porosity than theother side), and good strength, for example burst strength. Also, it hasbeen discovered that more preferred processes do not affect thedifferential porosity of the tela submucosa collagen biomaterial, whichultimately affects the level of efficacy of this tissue implant. Forexample, the tissue is not necessarily. treated with a crosslinkingagent or a material that disrupts the porosity or inherent, nativestructure of the collagen biomaterial. Moreover, when hydrogen peroxideis employed, the biomaterial as a whole has greater porosity as well asa higher oxygen content. This helps to ensure the absence ofcontaminants e.g., endotoxins, pyrogens, and the like.

Preferred collagen-based matrices of the invention, preferablysubmucosa-containing matrices, are also characterized by the lowcontaminant levels set forth in Table 1 below, each contaminant leveltaken individually or in any combination with some or all of the otherdisclosed contaminant levels. The abbreviations in Table 1 are asfollows: CFU/g=colony forming units per gram; PFU/g=plaque forming unitsper gram; pg/mg=micrograms per milligram; ppm/kg=parts per million perkilogram; and EU/g=endotoxin units per gram.

TABLE 1 FIRST SECOND THIRD PREFERRED PREFERRED PREFERRED FEATURE LEVELLEVEL LEVEL ENDOTOXIN <12 EU/g <10 EU/g <5 EU/g BIOBURDEN <2 CFU/g <1CFU/g <0.5 CFU/g FUNGUS <2 CFU/g <1 CFU/g <0.5 CFU/g NUCLEIC <10pg/mg_(i) <5 Ng/mg <2 pg/mg ACID VIRUS <500 PFU/g <50 PFU/g <5 PFU/gPROCESSING <100,000 ppm/kg <1,000 ppm/kg <100 ppm/kg AGENT

Even more preferred collagen-based matrices 10 of the invention containan endotoxin level of less than 1 EU/g, and most preferably less than0.5 EU/g.

Purified collagen-based matrices according to the present invention canbe processed in a number of ways, to provide collagenous matrices usefulboth in vitro and in vivo. For example, the submucosa can be configuredto provide tissue grafts useful in vascular applications, e.g. asgenerally described in U.S. Pat. Nos. 2,127,903 and 4,902,508.

The tela submucosa of the invention possesses mechanical propertieshighly desirable for tissue graft materials in vascular applications,including low porosity index, high compliance, and a high burststrength. One skilled in the art will appreciate that the preferredtissue graft material will be of low enough porosity to preventintraoperative hemorrhage and yet of high enough porosity to allowextension of a newly-developed vasa vasorum through the graft materialto nourish the neointimal and luminal surface.

Tela submucosa tissue of the present invention can also be processed toprovide fluidized compositions, for instance using techniques asdescribed in U.S. Pat. No. 5,275,826. In this regard, solutions orsuspensions of the tela submucosa can be prepared by comminuting and/ordigesting the tela submucosa with a protease (e.g. trypsin or pepsin),for a period of time sufficient to solubilize the tissue and formsubstantially homogeneous solution. The submucosa starting material isdesirably comminuted by tearing, cutting, grinding, shearing or thelike. Grinding the submucosa in a frozen or freeze-dried state isadvantageous, although good results can be obtained as well bysubjecting a suspension of pieces of the submucosa to treatment in ahigh speed blender and dewatering, if necessary, by centrifuging anddecanting excess waste. The comminuted tela submucosa can be dried, forexample freeze dried, to form a powder. Thereafter, if desired, thepowder can be hydrated, that is, combined with water or buffered salineand optionally other pharmaceutically acceptable excipients, to form afluid tissue graft composition, e.g. having a viscosity of about 2 toabout 300,000 cps at 25° C. The higher viscosity graft compositions canhave a gel or paste consistency.

Fluidized tela submucosa of this invention finds use as an injectableheterograft for tissues, for example, bone or soft tissues, in need ofrepair or augmentation most typically to correct trauma ordisease-induced tissue defects. The present fluidized submucosacompositions are also used advantageously as a filler for implantconstructs comprising, for example, one or more sheets of tela submucosaformed into sealed (sutured) pouches for use in cosmetic ortrauma-treating surgical procedures.

In one illustrative preparation, tela submucosa prepared as describedherein is reduced to small pieces (e.g. by cutting) which are charged toa flat bottom stainless steel container. Liquid nitrogen is introducedinto the container to freeze the specimens, which are then comminutedwhile in the frozen state to form a coarse tela submucosa powder. Suchprocessing can be carried out, for example, with a manual arbor presswith a cylindrical brass ingot placed on top of the frozen specimens.The ingot serves as an interface between the specimens and the arbor ofthe press. Liquid nitrogen can be added periodically to the telasubmucosa specimens to keep them frozen.

Other methods for comminuting tela submucosa specimens can be utilizedto produce a tela submucosa powder usable in accordance with the presentinvention. For example, tela submucosa specimens can be freeze-dried andthen ground using a manual arbor press or other grinding means.Alternatively, tela submucosa can be processed in a high shear blenderto produce, upon dewatering and drying: a tela submucosa powder.

Further grinding of the tela submucosa powder using a prechilled mortarand pestle can be used to produce a consistent, more finely dividedproduct. Again, liquid nitrogen is used as needed to maintain solidfrozen particles during final grinding. The powder can be easilyhydrated using, for example, buffered saline to produce a fluidizedtissue graft material of this invention at the desired viscosity.

To prepare another preferred fluidized material, a tela submucosa powdercan be sifted through a wire mesh, collected, and subjected toproteolytic digestion to form a substantially homogeneous solution. Forexample, the powder can be digested with 1 mg/ml of pepsin (SigmaChemical Co., St. Louis Mo.) and 0.1 M acetic acid, adjusted to pH 2.5with HCl, over a 48 hour period at room temperature. After thistreatment, the reaction medium can be neutralized with sodium hydroxideto inactivate the peptic activity. The solubilized submucosa can then beconcentrated by salt precipitation of the solution and separated forfurther purification and/or freeze drying to form a protease-solubilizedintestinal submucosa in powder shape.

Fluidized tela submucosa compositions of this invention find wideapplication in tissue replacement, augmentation, and/or repair. Thefluidized submucosal compositions can be used to induce regrowth ofnatural connective tissue or bone in an area of an existent defect. Byinjecting an effective amount of a fluidized submucosa composition intothe locale of a tissue defect or a wound in need of healing, one canreadily take advantage of the biotropic properties of the telasubmucosa. Interestingly, fluidizing SIS by comminution or enzymaticdegradation does not result in any appreciable loss of biotropicactivities, as shown in U.S. Pat. No. 5,275,826.

It is also possible to shape large surface area constructs by combiningtwo or more tela submucosa segments of the invention, for instance usingtechniques as described in U.S. Pat. No. 2,127,903 and/or InternationalPublication No. WO. 96/32146, dated 17 Oct. 1996, publishingInternational Application No. PCT/US96/04Z71, filed 5 Apr. 1996. Thus, aplurality of tela submucosa strips can be fused to one another, forexample by compressing overlapping areas of the strips under dehydratingconditions, to form an overall planar construct having a surface areagreater than that of any one planar surface of the individual stripsused to shape the construct. Shapes can be made by using sutures,staples, biocompatible adhesives such as collagen binding pastes, ordehydrating overlapping structures then heating the structure asdescribed in U.S. Pat. No. 3,562,820.

The tele submucosa powder can be used alone, or in combination with oneor more additional bioactive agents such as physiologically compatibleminerals, growth factors, antibiotics, chemotherapeutic agents, antigen,antibodies, enzymes and hormones. Preferably, the powder-form implantwill be compressed into a predetermined, three-dimensional shape, whichwill be implanted into the bone region and will substantially retain itsshape during replacement of the graft with endogenous tissues.

Tele submucosa of the invention can also be used as a cell growthsubstrate, illustratively in sheet, paste or gel shape in combinationwith nutrients which support the growth of the subject cells, e.g.eukaryotic cells such as endothelial, fibroblastic, fetal skin,osteosarcoma, and adenocarcinoma cells (see, e.g. InternationalPublication No. WO 96/24661 dated 15 Aug. 1996, publishing InternationalApplication No. PCT/US96/01842 filed 9 Feb. 1996. In preferred forms,the tela submucosa substrate composition will support the proliferationand/or differentiation of mammalian cells, including human cells.

The inventive tela submucosa can also serve as a collagenous biomaterialin compositions for producing transformed cells, (see, e.g.,International Publication No. WO 96/25179 dated 22 Aug. 1996, publishingInternational Application No. PCT/US96/02136 filed 16 Feb. 1996; andInternational Publication No. WO 95/22611 dated 24 Aug. 1995, publishingInternational Application No. PCT/US95/02251 filed 21 Feb. 1995). Suchcompositions for cell transformation will generally include purifiedtela submucosa of the present invention, for example in fluidized orpaste shape as described in U.S. Pat. No. 5,275,826, in combination witha recombinant vector (e.g. a plasmid) containing a nucleic acid sequencewith which in vitro or in vivo target cells are to be geneticallytransformed. The cells targeted for transformation can include, forexample, bone progenitor cells. In addition to the medicaments describedabove, the biomaterial may also be used as a vehicle for genetictherapy, in which exogenous nucleic acids are disposed on the graft 10.As such upon implantation, local tissue takes up the nucleic acids andthus gene transfer occurs. The process for this is described in U.S.Pat. No. 5,763,416 to Bonadio et al., the disclosure of which isexpressly incorporated by reference. As such, the graft 10 can comprisea biomaterial having an extension 14, an aperture 16, and an exogenousnucleic acid disposed thereon.

In order to promote a further understanding of the present invention andits features and advantages, the following specific Examples areprovided. It will be understood that these specific Examples areillustrative, and not limiting, of the present invention.

Example 1

Thirty feet of whole intestine from a mature adult hog is rinsed withwater. This material is then treated in a 0.2% by volume peracetic acidin a 5% by volume aqueous ethanol solution for a period of two hourswith agitation. The tela submucosa layer is then delaminated in adisinfected casing machine from the whole intestine. The delaminatedtela submucosa is rinsed four (4) times with sterile water and testedfor impurities or contaminants such as endotoxins, microbial organisms,and pyrogens. The resultant tissue was found to have essentially zerobioburden level. The tela submucosa layer separated easily andconsistently from the whole intestine and was found to have minimaltissue debris on its surface.

Example 2

A ten foot section of porcine whole intestine is washed with water.After rinsing, this section of tela submucosa intestinal collagen sourcematerial is treated for about two and a half hours in 0.2% peraceticacid by volume in a 5% by volume aqueous ethanol solution withagitation. Following the treatment with peracetic acid, the telasubmucosa layer is delaminated from the whole intestine. The resultanttela submucosa is then rinsed four (4) times with sterile water. Thebioburden was found to be essentially zero.

Example 3

A small section of the tela submucosa intestinal collagen material wassubcutaneously implanted in a rat. Within 72 hours, significantangiogenesis was observed.

Example 4

Two sections of small intestine are processed by differing methods. Thefirst section is rinsed in tap water, disinfected for 2 hours in a 5% byvolume aqueous ethanol solution comprising 0.2% by volume peraceticacid, pH approximately 2.6, delaminated to the tela submucosa, rinsed inpurified water, divided into two samples and rapidly frozen. The secondsection is rinsed in tap water, delaminated to the tela submucosa,rinsed in purified water, placed in a 10% neomycin sulfate solution for20 minutes (as described in U.S. Pat. No. 4,902,508), rinsed in purifiedwater, divided into two samples and rapidly frozen. The fourabove-prepared samples are tested for bioburden and endotoxin levels.The first two samples each have bioburdens of less than 0.1 CFU/g andendotoxin levels of less than 0.1 EU/g. The second two samples haverespective bioburdens of 1.7 CFU/g and 2.7 CFU/g and respectiveendotoxin levels of 23.9 EU/g and 15.7 EU/g.

Example 5

Three sections of small intestine are processed by differing methods.The first is rinsed in tap water, disinfected for 2 hours in a 5% byvolume aqueous ethanol solution comprising 0.2% by volume peraceticacid, pH about 2.6, delaminated to the tele submucosa, rinsed inpurified water, and rapidly frozen. The second is rinsed in tap water,delaminated to the tele submucosa, rinsed in purified water, disinfectedaccording to the methods of Example 1 in U.S. Pat. No. 5,460,962(treatment for 40 hours in a 0.1% by volume aqueous solution ofperacetic acid, buffered to pH 7.2), and rapidly frozen. The third isrinsed in tap water, delaminated to the tele submucosa, rinsed inpurified water, disinfected according to the methods of Example 2 inU.S. Pat. No. 5,460,962 (treatment in 0.1% by volume peracetic acid inhigh salt solution, buffered to pH 7.2), and rapidly frozen. All threesamples were tested for endotoxins. The endotoxin levels were <0.14 EU/gfor the first sample, >24 EU/g for the second sample, and >28 EU/g forthe third sample.

Example 6

Two sections of porcine small intestine were infected with 7×10⁶ plaqueforming units (PFU) of virus. Both were exposed to a 0.18% peraceticacid, 4.8% aqueous ethanol solution at a nine-to-one weight ratio ofsolution to material. A first sample was immersed in this solution for 5minutes; the second was immersed for 2 hours. The material processed for5 minutes exhibited 400 PFU per gram of material. The material processedfor 2 hours exhibited zero PFU per gram of material.

Example 7

Purified tele submucosa, prepared as described herein, was tested todetermine its nucleic acid content. Four samples of material weighing 5mg each were subjected to DNA/RNA extraction as detailed in the DNA/RNAIsolation Kit by Amersham Lifescience Inc., Arlington Heights, Ill.Nucleic acid quantitation was performed by spectrophotometricdetermination of solution optical densities at 260 nm and 280 nm. Theaverage nucleic acid content was 1.9±0.2 mg per milligram of material.

Small intestinal submucosa, prepared as described by U.S. Pat. No.4,902,508, was tested to determine its nucleic acid content. Foursamples of material weighing 5 mg each were subjected to DNA/RNAextraction as detailed in the DNA/RNA Isolation Kit by Amersham. Nucleicacid quantitation was performed by spectrophotometric determination ofsolution optical densities at 260 nm and 280 nm. The average nucleicacid content was 2.4±0.2 mg per milligram of material.

Example 8

Sections of tela submucosa prepared according to the methods describedherein were sent to an independent testing laboratory (NamSA, Inc.,Northwood, Ohio) for biocompatibility testing as described in thestandard ISO 10993. The samples were tested for USP Acute SystemicToxicity, USP Intracutaneous Toxicity, Cytotoxicity, LAL Endotoxin,material-mediated Pyrogenicity, Direct Contact Hemolysis, and PrimarySkin Irritation. The samples passed all tests, indicating that thematerial is biocompatible.

Example 9

Using the procedure set forth in U.S. Pat. No. 5,460,962, two sampleswere analyzed. The first Kemp sample indicated an endotoxin levelgreater than 24 endotoxin units per gram and the second Kemp sampleindicated an endotoxin level greater than 28 endotoxin units per gram.Thus, when using the procedure set forth in Kemp '962, the endotoxinlevels fall outside the biocompatibility levels.

Example 10

Using the procedures set forth in U.S. Pat. Nos. 4,902,508 and 5,372,821issued to Badylak, the endotoxin level shown ranges as high as 23.9endotoxin units per gram of sample. This falls outside the permissiblerange and thus does not meet the criteria of biocompatibility as definedabove. The invention, prepared in the above prescribed manner ofdisinfection first then delamination, was observed to have an endotoxinlevel of less than 12 endotoxin units per gram, and more particularly,reported an endotoxin level of less than 5 endotoxin units per gram.Thus, the material of the present invention is biocompatible as definedabove.

With particular reference to FIGS. 1 and 2, shown in FIG. 1 is the graft10 in partially folded condition, and shown in FIG. 2 is the graft 10 inflat sheet form. Graft 10 comprises a first biomaterial 12 with at leastone extension 14, with most likely a plurality of extensions 14.Extension 14 includes a tab, a projection, an insertion, or a maleportion of a lock. Also provided on graft 10 first biomaterial 12 is atleast one aperture 16, with most likely a plurality of apertures 16.Aperture 16 includes a slit, a hole, an orifice, or a receiving member.Extension 14 is generally shaped so that extension 14 can be insertedinto aperture 16. Extension 14 need not, however, be the same size ofaperture 16. Extension 14 need only be shaped in such a way as to insertinto aperture 16. To this end, extension 14 may be rectangular, mushroomshaped, or club shaped. In addition, the extension 14 and aperture 16include the well-known male-female locking mechanisms.

In one embodiment of the invention, extension 14 is larger or wider thataperture 16. In this manner, the surplusage of extension 14 materialfitting through or in aperture 16 causes the extension 14 to not comeout or get pulled out of aperture 16. In this regard extension 14 doesnot get pulled out of aperture 16 causing extension 14 to be relatively“locked” into place.

Extension 14 is moved into proximity of aperture 16, usually by foldingthe fist biomaterial 12 sheet in direction 17. That is, when the sheetis flat lying on a table, the inside wall 18 of graft 10 is the surfacefacing up, thus forming inside wall surface 20. The exterior surface 22of extension 14 is the surface adjacent to the table. As such, graft 10is formed by grasping the sides and bringing them into proximity of eachother direction 17. As such, extension 14 has a leading edge 24 andleading edge 24 inserts into aperture 16. If a series of extensions 14are present; then selective extensions 14 can be inserted into oneaperture 16 or a plurality of apertures 16.

As further shown in FIGS. 2 and 3, aperture 16 can be along the edge ofthe sheet or at some distance 19, an extra amount or surplus material 26is formed. This material can be the same material as the sheet or can bea second material 26.

As shown in FIG. 4, leading edge 24 is inserted into aperture 16. As thesheet lies flat on the table, the inside wall 18 is partially seen inbetween the plurality of extensions 14. The exterior surface 22 ofextension 14 is on the top side or opposite side to the inside wall 18.

As shown in FIGS. 5 and 6, leading edge 24 of extension 14 is insertedinto and through aperture 16. As leading edge 24 is pulled through theaperture, a tube is created at that point, thus creating lumen 27. Assuccessive extensions 24 are pulled through the aperture, the rest ofthe tube is created, creating an extended lumen 27. The “seam” resemblesthe familiar dove-tail join characteristically found in wood-workingfurniture assembly.

As shown in FIG. 7, as the successive extensions 14 are pulled through25 the apertures 16, the surplus material 26 can be folded or rolledaround the newly forming tube. In this regard, the surplus material 26can be folded or rolled in such a manner as to cover or overlap theaperture 16-extension 14 junction 25. This way, upon completion of thegraft 14, surplus material 26 will provide a seal over the aperture 16,and more particularly, over junction 25. The more times that surplusmaterial 26 can wrap around the tube, the greater the structuralintegrity that is present. In addition, the existence of seepage orleakage is dramatically reduced.

FIG. 8 shows another embodiment of the present invention. To provideextra leak protection or to provide medicaments, an intermediate layer28 can be strategically placed over the aperture 16-extension 14junction 25. The intermediate layer 28 or outer layer 30 comprises atleast one of the submucosal tissue, mucosal tissue, serosal tissue,collagen, partially collagenous biomaterial, elastin,polytetrafluoroethylene, polyester, stainless steel, DACRON (R),ORLON(R), FORTISAN(R), nylon, polypopylene, polyglatin 910, polyglycolicacid, pericardium, dura tissue, facia lata, a biocompatible material,polymers, co-polymers, polypyrramidole, a synthetic material, and anycombination or part thereof. Thus, in its simplest but non exclusiveembodiment, the graft 10 can comprise a biological tissue for theinitial sheet, a biological tissue for the surplus material 26, abiological tissue for the intermediate layer 28, and a biological tissuefor outer layer 30. However, any combination of materials may be used.For example, the initial sheet of first biomaterial 12 and surplusmaterial 26 may comprise biological tissue, the intermediate layer 28may comprise DACRON(R), and the outer layer 30 may comprise a second ordifferent biological tissue. Therefore, specifically contemplated isthat the graft 10 may include at least 4 types of materials. Also, it ispossible to include a plurality of various layers as outer layers 30 tovary the composition, structural integrity, therapeutic value, or thelike. For example, if long term medication is needed in the area,intermediate layer 28 may be treated with medicaments to provide a longterm application. Similarly, outer layer 28 may be treated withmedicaments to facilitate treatment to the adjacent or systemic tissues.In addition, any layer may completely or partially surround the graft10, or completely or partially surround any inner layers. As mentionedherein, any layer, including the first biomaterial 12 itself, may betreated with medicaments to provide therapy to an afflicted area.

FIG. 9 shows another embodiment of the present invention. Apertures 16need not be in a line equidistant from the sheet edge. In fact, theapertures may be staggered or randomly placed to provide a varying shapeor lumen size of the overall graft 10. By calculating the variousconfigurations, one could create a tube with a gradually increasing ordecreasing lumen size.

FIG. 10 shows another embodiment of the present invention. In this case,graft 10 is provided with a woven outer layer 30. This outer layer 30,as with intermediate layer 28 (as shown in FIG. 8), comprise abiocompatible material such as submucosal tissue, mucosal tissue,serosal tissue, collagen, partially collagenous biomaterial,polytetrafluoroethylene, polyester, stainless steel, DACRON(R),ORLON(1=1), FORTISAN(R), nylon, polypropylene, polyglactin 910,polyglycolic acid, pericardium, dura tissue, facia lata, a biocompatiblematerial, polymers, polypyrramidole, co-polymers, a synthetic material,and any combination or part thereof.

FIG. 11 shows a section of the extensions 14. This embodimentparticularly uses a retainer 32 to relatively “lock” the extension 14into place after insertion into aperture 16. In this embodiment of theinvention, the retainer 32 is actually part of the initial sheet.However, specifically contemplated are other retainers 32, such aslocks, sutures, adhesives, staples, suture wire on the ablumenal side orother well known methods for creating a retainer 32. Any method oflocking two sections of tissue together is specifically contemplatedherein. In this non-limiting embodiment, the dotted lines of FIG. 11indicate where the sheet is cut to form each extension 14. Thus the“locking” mechanisms are not entirely within the lumen.

FIG. 12 shows a partial section of one extension 14. To form a retainer32, extension 14 is folded along the dotted lines in the direction shownto create a partial retainer 34. The extension 14 has a centralextension 36, the central extension having a width designated X. Acrossthe sheet is aperture 16, with aperture 16 having a width of X also.However, the widths need not be the same. When extension 14 is foldedalong the dotted lines shown to create width X of central extension 36,it facilitates insertion into aperture 16. When extension 14 is pulledthrough the aperture 16, the user can then unfold each partial retainer34. Since each partial retainer has a width Y, the combined widths ofX+Y+Y is greater than width X of the aperture 16. Thus, the extension 14cannot easily be pulled back through the aperture 16 into the unfoldedsheet configuration. Similarly, retainer 32 comprises adhesives, partialretainers, sutures, staples, or any other structure capable of securingextension 14 such that it retards the removal of extension 14 from, orthrough, the aperture 16.

FIG. 13 shows the unfolded position of extension 14, partial retainers34, and forming the retainer 32. In this regard, the arrows indicatethat each partial retainer 34 is unfolded. Evidently, the width ofaperture 16 (designated as X) is the same as the width of the centralextension 36 (also designated as X), but the width of extension 14 isX+Y+Y and thus, extension 14 will not easily be pulled out of aperture16. However, the width of extension 14 need not be the same as the widthof aperture 16.

Returning now to FIG. 8, once the graft 10 is made with intermediatelayers 28 or outer layers 30, where appropriate, the graft 10 is thentreated. Treatment includes freeze drying the graft 10. By freeze dryingthe graft 10, the layers are pressed further together thus providingextra leakage protection and increased structural integrity. The problemassociated with other grafts made from sheets is that the inner seam onthe lumenal surface 18, 20 often “hangs” down or protrudes into thelumen. That is, the layers begin to peel away and separate from eachother. This poses a serious problem as a potential source ofthrombogenesis. However, the present invention solves this problem byproviding an “interrupted” or discontinuous seam in the lumen 27. Inaddition, after freeze drying the interface is sealed. Microscopicexamination provides no visible evidence of the extensions 14, theapertures 16, or the junction 25. This is because the interface consistsof adjacent extensions and apertures engaging each other. Thus, thegraft 10 is more impermeable to leakage and provides better protection.It reduces the thrombogenesis since nothing exists to hang down into thelumen 27.

Other embodiments include using a multi-laminate sheet with which tostart. For example, several collagen sheets may be used to increasestructural integrity. In another example, multiple mucosal sheets can beused, as described in U.S. Pat. No. 5,885,619 to Patel et al., thedisclosure of which is expressly incorporated by reference; or describedin U.S. Pat. No. 5,755,791 to Whitson et al., the disclosure of which isexpressly incorporated by reference.

Returning now to FIGS. 8 and 10, yet another embodiment of the presentinvention includes a marker 38. Marker 38 is used to initially mark thelocation of the junction 25. In this regard, during the manufacturingprocess, the marker 38 is visible and will indicate to the user thelocation of junction 25. In yet another embodiment of the presentinvention, the graft or any individual constituent thereof may alsocontain a drug or chemical coating 40. For example, any layer orconstituent thereof may contain a heparin coating to prevent thrombosis.In yet another embodiment of the present invention, the graft can bemade radiographically visible by either making the retainer or a markerradiovisible, or making the graft radiovisible via heavy metal powders42, such as tantalum or barium.

With reference now to FIG. 3A, shown is another embodiment of thepresent invention. Graft 10′ shown in FIG. 3A is similar to the graftshown in FIG. 3, except that extensions 14′ and apertures 16′ occur onalternating sides of the sheet. Graft 10′ is also similarly assembledinto tube form, except that the folding of the extensions 14′ (inwardlyalong dotted lines as indicated by arrows) and their insertion intocorresponding apertures 16′ can be carried out in alternating fashionand in alternating directions as the tube is assembled.

Shown in FIG. 14 is another embodiment of the invention. Graft 50 forforming a tubular structure is formed from a sheet of biomaterial orother similar biocompatible material that includes extensions 52 offsetwith respect to and configured to interleave with extensions 53occurring on the opposite side of graft 50. In this fashion, seams willbe formed at the contact points of non-edge portions 54 of extensions 52and edge portions 55 occurring between extensions 53 on the oppositeside of graft 50, and at the contact points of non-edge portions 56 ofextensions 53 and edge portions 57 occurring between extensions 52. Inthis manner, multiple, relatively smaller seam structures will bepresented on the inner lumen wall of the assembled tubular graft 50. Aswill other grafts on the invention, extensions 52 and 53 may optionallybe of sufficient length to extend around the circumference of theassembled tube at least one time so as to overlap the multiplelongitudinal seams created at the contact of portions 54 and 55, andportions 56 and 57, respectively. As in other constructs of theinvention, a suitable biocompatible and/or bioresorbable bonding agentmay be applied to surfaces of extensions 52 and 53 or in other locationson the tube to facilitate integrity of the tube, and/or attachments ofthe layers may be achieved by crosslinking or other suitable methods.

FIGS. 15 and 16 illustrate another embodiment of the invention. Graft 60is somewhat similar in design to graft 50 of FIG. 14, except that sheet61 of biomaterial or other similar biocompatible material has extensions62 and 64 formed by appropriate cuts in the sheet so as to configure tointerleaving extensions upon folding each extension atop itself (e.g. byfolding along dotted lines 63 and 65 in FIG. 15 in the direction of thearrows). In this fashion, a construct having folded extensions 62 and 64configured to interleave as folded is created (see FIG. 16), which canthen be assembled to a tube similar to graft 50 of FIG. 14. However,after interleaving the extensions 62 and 64, they may be unfolded so asto create an overlapping set of interleaved extensions, providing forexample additional overlapped surface area for bonding, crosslinkage orother attachment, and additional resistance to separation.

FIGS. 17 and 18 show another embodiment of the invention, in which abutt joint is utilized to create a tubular graft having a lumen wallfree of any seam edge. In particular, tubular graft 70 formed especiallyof a biomaterial such as a collagenous sheet 71, is formed byconfiguring the sheet to a tube and creating a butt joint 72 to create afluid-tight seal of the tube. Butt joint 72 is created by contactbetween portions of the same side of the sheet 71 and forms alongitudinal seam along the tube, but presents no edge of the originalsheet. Butt joint 72 can be created using any suitable bonding or otherattachment method, including for example the use of dehydration bonding(e.g. in the case of a collagenous material), a biocompatible and/orbioresorbable bonding agent, crosslinking, or the like.” The biomaterialsheet may be of sufficient size to provide a length of surplus sheetwhich can be wrapped about the formed tube (e.g. in the direction ofarrow, FIG. 17) so as to overlap the butt joint 72 at least once asillustrated in FIG. 18 or multiple times if desired. Further, additionallayers of material may be provided around graft 70 to form a finishedconstruct if desired. For example, additional layers of biomaterial maybe provided and/or a tube of synthetic material as described herein maybe provided around the outside of graft 70 in forming the finishedtubular construct. Graft constructs having one or more butt jointscreating a lumen seal can be advantageous, for example, where the sheetstarting material has a sidedness, wherein presentation of one side ofthe material in the lumen (e.g. at an edge or upon partial detachment ofa seam) of the graft is desirably avoided. For example, an isolatedcollagenous layer having differing physical or chemical properties onone side as compared to the other, for example small intestinalsubmucosa, may be used with preference with a certain side forming thelumen of the graft (e.g. the lumina) side in the case of smallintestinal submucosa), and the other side (e.g. abluminal side)occurring as the outer wall. In the case of a butt jointed tubulargraft, only additional amounts of the originally-presented side of thesheet will potentially be exposed to the lumen of the graft. Forexample, in a butt-jointed tubular graft having a lumen formed of theluminal side small intestinal submucosa, no edge of the submucosa willbe exposed to the lumen, and only additional luminal side portions ofthe submucosa (and not abluminal portions) would be exposed to the lumenof the tubular graft if some detachment of the seam occurred.

As discussed herein, tubular grafts of the invention may have layers,for example biomaterial layers such as collagenous layers, attached toone another to facilitate integrity of the construct. Attachment oflayers may be achieved for example utilizing dehydration bonding of thelayers (e.g. with freeze or vacuum drying), crosslinking with suitablecrosslinking agents such as glutaraldehyde, formaldehyde, or the like,and/or using resorbable or non-resorbable biocompatible bonding agentssuch as fibrin glue, cyanoacrylates, chitin or chitosan based bondingagents, elastin containing bonding agents, or any combination thereof.Other techniques such as suturing may also be used alone or incombination with other attachment Methods.

Graft constructs of the invention may be terminally sterilized usingwell known sterilization techniques such as radiation, ethylene oxide,or gas plasma.

Since the graft is intended to be a medical device, the graft willlikely be packaged in some sterile packaging or packaged then terminallysterilized. Thus, the medical device may comprise a graft constructidentified above, including a package with the package and graft beingterminally sterilized.

The compositions and methods disclosed herein are only intended to beexamples of the present invention. It will be apparent to those skilledin the art that modifications may be made without undue experimentationand it is contemplated that any of these modifications are within thescope of the appended claims. All publications cited herein areindicative of the level of skill possessed by those working in the artand each such publication is hereby incorporated by reference in itsentirety.

1. A medical device, comprising: a compliant sheet material harvestedfrom a collagenous tissue source, the sheet material providing a firstextension, and further having a first aperture extending through thesheet material, the first extension inserted through the first aperturesuch that a surface portion of the first extension overlaps anunderlying surface of the sheet material to provide a multi-laminatedregion of said compliant sheet material, and wherein said surfaceportion is bonded to said underlying surface.
 2. The medical device ofclaim 1, wherein said first extension includes a portion having a widthgreater than a maximum width of said first aperture.
 3. The medicaldevice of claim 1, further comprising a second material disposed on thecompliant sheet material.
 4. The medical device of claim 3, furthercomprising an intermediate layer disposed under the second material. 5.The medical device of claim 4, wherein at least one of the secondmaterial and the intermediate layer comprises at least one of asubmucosal tissue, mucosal tissue, collagen, partially collagenousbiomaterial, polytetrafluoroethylene, polyester, stainless steel,DACRON, ORLON, FORTISAN, nylon, polypropylene, polyglactin 910,polyglycolic acid, pericardium, dura tissue, facia lata, a biocompatiblematerial, a synthetic material, polymers, co-polymers, and anycombination or part thereof.
 6. A medical device, comprising: a) a tubehaving a lumen extending therethrough and including a wall formed with acompliant sheet material harvested from a collagenous tissue source, thesheet material having a first side forming a surface adjacent said lumenand a second side forming an external surface of said tube; b) whereinthe sheet material has a plurality of extensions and a plurality ofapertures, with each of the plurality of apertures providing an openingextending through the sheet material, c) wherein each one of saidplurality of extensions has a first extension portion received through acorresponding one of said plurality of apertures so as to overlie anunderlying layer of material, wherein a surface of said first extensionportion conforms and is bonded to the underlying layer of material byone or more of dehydrothermal bonding, crosslinking, or bonding with aresorbable or non-resorbable biocompatible bonding agent.
 7. The medicaldevice of claim 6, wherein the first extension portion of at least oneof said plurality of extensions has a width greater than a maximum widthof the corresponding aperture through which the first extension portionis received.
 8. The medical device of claim 6, wherein the firstextension portion of at least one of said plurality of extensionsincludes a retainer.
 9. The medical device of claim 6, wherein saidcompliant sheet material comprises a collagenous extracellular matrixmaterial.
 10. The medical device of claim 9, wherein said collagenousextracellular matrix material comprises submucosal tissue.
 11. A medicaldevice, comprising: a tube formed from a compliant sheet material, thetube having a lumen having a lumen wall, said lumen wall free from anycontinuous seam edge traversing the entire length of the tube, and saidlumen wall including at least one multi-layer region in which a firstportion of said sheet material is bonded to an underlying second portionof said sheet material.
 12. The medical device of claim 11, wherein thelumen wall presents a plurality of longitudinal seam edges.
 13. Themedical device of claim 12, wherein said seams are formed byintersections of edge portions of said sheet of biomaterial and non-edgeportions of said sheet of biomaterial.
 14. The medical device of claim13, wherein said edge portions are formed at apertures in said sheet ofbiomaterial.
 15. The medical device of claim 13, wherein said tubecomprises a plurality of extensions extending through a plurality ofcorresponding apertures in said biomaterial.
 16. The medical device ofclaim 13, wherein said edge portions are formed at a perimeter of saidsheet of biomaterial.
 17. The medical device of claim 16, wherein saidtube comprises a plurality of interleaving extensions of saidbiomaterial.
 18. The medical device of claim 11, wherein said tubecomprises a seam formed by a butt joint.
 19. A medical device,comprising: a tube having a lumen extending therethrough and including awall formed with a compliant sheet material harvested from a collagenoustissue source, the sheet material having a first side forming a surfaceadjacent said lumen and a second side forming an external surface ofsaid tube, wherein said wall has a discontinuous seam.
 20. The medicaldevice of claim 19, wherein said discontinuous seam includes a pluralityof seams each formed by edge portions of said biomaterial in contactwith non-edge portions of said biomaterial.